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Studies in Saccharomyces cerevisiae indicate the histone variant H2A.Z is deposited at promoters by the chromatin remodeling protein Swr1 and plays a critical role in the regulation of transcription. In higher eukaryotes, however, little is known about the distribution, method of deposition, and function of H2A.Z at promoters. Using biochemical studies, we demonstrated previously that SRCAP (SNF-2-related CREB-binding protein activator protein), the human ortholog of Swr1, could catalyze deposition of H2A.Z into nucleosomes. To address whether SRCAP directs H2A.Z deposition in vivo, promoters targeted by SRCAP were identified by a chromatin immunoprecipitation (ChIP)-on-chip assay. ChIP assays on a subset of these promoters confirmed the presence of SRCAP on inactive and active promoters. The highest levels of SRCAP were observed on the active SP-1, G3BP, and FAD synthetase promoters. Detailed analyses of these promoters indicate sites of SRCAP binding overlap or occur adjacent to the sites of H2A.Z deposition. Knockdown of SRCAP levels using siRNA resulted in loss of SRCAP at these promoters, decreased deposition of H2A.Z and acetylated H2A.Z, and a decrease in levels of SP-1, G3BP, and FAD synthetase mRNA. Thus, these studies provide the first evidence that SRCAP is recruited to promoters and is critical for the deposition of H2A.Z.

The Snf-2-related CREB-binding protein activator protein (SRCAP) serves as a coactivator for a number of transcription factors known to interact with CBP. Swr1, the closest Saccharomyces cerevisiae ortholog of SRCAP, is a component of the chromatin remodeling complex SWR-C, which catalyzes exchange of the histone variant H2A.Z into nucleosomes. In this report, we use a combination of conventional chromatography and anti-SRCAP immunoaffinity chromatography to purify a native human SRCAP complex with a polypeptide composition similar to that of SWR-C, and we show for the first time that this SRCAP-containing complex supports ATP-dependent exchange of histone dimers containing H2B and H2A.Z into mononucleosomes reconstituted with recombinant H2A, H2B, H3, and H4. Our findings, together with previous evidence implicating H2A.Z in transcriptional regulation, suggest that SRCAP's coactivator function may depend on its ability to promote incorporation of H2A.Z into chromatin.

The putative ATPase chromatin-remodeling machine SRCAP was identified in a yeast two-hybrid protein screen by interaction with the histone acetylase CBP. SRCAP is implicated in the transcriptional coactivation of cyclic AMP- and steroid-dependent promoters, but no natural chromosomal targets for SRCAP regulation have been identified. DOM is the unique SRCAP homolog in Drosophila melanogaster. The goal of this study was to test whether SRCAP is a functional homolog of DOM and to identify potential activities and targets of SRCAP in vivo. We show that human SRCAP complements recessive domino mutant phenotypes. This rescue depends on an intact ATPase homology domain. SRCAP colocalizes extensively with DOM on Drosophila polytene chromosomes and is recruited to sites of active transcription, such as steroid-regulated loci, but not to activated heat shock loci. We show that SRCAP recruits Drosophila CBP to ectopic chromosomal sites, providing the first evidence to suggest that SRCAP and CBP interact directly or indirectly on chromosomes. We show that DOM is a Notch pathway activator in Drosophila and that wild-type SRCAP-but not an ATPase domain mutant-can substitute for DOM in Notch-dependent wing development. We show that SRCAP potentiates Notch-dependent gene activation in HeLa cells. Taken together, these data implicate SRCAP and DOM in developmental gene activation.

To directly activate specific gene expression, the estrogen receptor (ER) must bind to estrogen receptor response elements (EREs) in the context of nucleosomes. In order to investigate the interaction of the ER with mononucleosomes, we developed a mononucleosome gel shift assay. A 164 bp high specific activity [ 32 P]probe DNA (32 bp consensus ERE with flanking regions separated by 23 nucleotides from an artificial nucleosome positioning sequence) was prepared. Nuclear extracts from MCF-7 cells or recombinant human ERα were incubated with the labeled ERE ± excess ERE. A retarded band was seen which was completely obliterated with excess ERE, confirming the specificity of binding. This probe was then used to make reconstituted mononucleosomes by sequential dilution of a high salt histone preparation. The nucleosomes were purified by sucrose density gradients and footprinting analysis was performed to demonstrate that the mononucleosomes were rotationally phased as seen by a periodic digestion pattern (10 bp) of the nucleosomes versus ERE. Nucleosomes were incubated with nuclear extracts containing ER or recombinant ERα. Dose dependence in the shift of the mononucleosomes with increasing concentrations of ER was observed. Specificity was demonstrated in experiments with excess ERE and anti-ER antibody. Footprinting analysis was also performed. We also determined that addition of high mobility group protein-2 (HMGB-2, a protein closely related to HMGB-1) with the ER increased the interaction of ER with mononucleosomes. These studies will allow us to address the interactions of ER with core histones containing a multiplicity of variants and modifications in nucleosomal structure.

The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275–2971). We report here that DBP inhibits the ability of full-length SrCap (1–2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP’s DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP.

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

The SNF2-related CBP activator protein, SrCap (pronounced “sir cap”), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.

Hepatitis C virus NS5A protein transcriptionally modulates cellular genes and promotes cell growth. NS5A is likely to exert its activity in concert with cellular factor(s). Using a yeast two-hybrid screen, we have demonstrated that NS5A interacts with the C-terminal end of a newly identified cellular transcription factor, SRCAP. The authenticity of this interaction was verified by a mammalian two-hybrid assay, in vitro pull-down experiment, and an in vivo coimmunoprecipitation assay in human hepatoma (HepG2) cells. An in vitro transient transfection assay demonstrated that SRCAP can efficiently activate transcription when recruited by the Gal4 DNA-binding domain to the promoter. However, down-regulation of p21 promoter activity by NS5A was enhanced following ectopic expression of SRCAP. Together these results suggest that the interaction of NS5A and SRCAP may be one of the mechanisms by which NS5A exerts its effect on cell growth regulation contributing to hepatitis C virus-mediated pathogenesis.

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.

CREB-binding protein (CBP) functions as a coactivator molecule for a number of transcription factors including CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. Although binding sites for these factors within CBP have been identified, the regions of CBP responsible for transcriptional activation are unknown. In this report, we show that the N-terminal half of CBP is sufficient for activation of CREB-mediated transcription and that this region contains a strong transcriptional activation domain (TAD). Both deletion of this TAD or sequestering of factors that the TAD binds using a squelching assay were found to greatly decrease the ability of CBP to activate CREB-mediated transcription. In vivo studies by others have shown that p300/CBP associates with TBP; using an in vitro approach, we show the N-terminal TAD binds TBP. We also examined the ability of the C terminus of CBP to activate transcription using GAL-CBP chimeras. With this approach, we identified two C-terminal TADs located adjacent to the c-Fos binding site. In previous studies, cAMP-dependent protein kinase A (PKA) increased the transcriptional activity of a GAL full-length CBP chimera in F9 cells, and of the C terminus in PC-12 cells. Here, we demonstrate that PKA also increased the ability of the N-terminal TADs of CBP to activate transcription in PC-12 but not F9 or COS-7 cells, suggesting that this PKA-responsiveness is cell type-specific.

The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.

The p400 and SRCAP (Snf2-related CBP activator protein) complexes remodel chromatin by catalyzing deposition of histone H2A.Z into nucleosomes. This remodeling activity has been proposed as a basis for regulation of transcription by these complexes. Transcript levels ofp21orSp1mRNAs after knockdown of p400 or SRCAP reveals that each regulates transcription of these promoters differently. In this study, we asked whether deposition of H2A.Z within specific nucleosomes by p400 or SRCAP dictates transcriptional activity. Our data indicates that nucleosome density at specificp21orSp1promoter positions is not altered by the loss of either remodeling complex. However, knockdown of SRCAP or p400 reduces deposition of H2A.Z∼50% into allp21andSp1promoter nucleosomes. Thus, H2A.Z deposition is not targeted to specific nucleosomes. These results indicate that the deposition of H2A.Z by the p400 or SRCAP complexes is not sufficient to determine how each regulates transcription. This conclusion is further supported by studies that demonstrate a SRCAPΔATPmutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP.

Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the protein CREB, which is activated as a result of phosphorylation by protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by protein kinase A binds specifically to a nuclear protein of M(r) 265K which we term CBP (for CREB-binding protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric protein to function as a protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.

Binding of ER to estrogen-regulated promoters has been extensively characterized in assays that utilize naked DNA as a template. In contrast, the factors that allow binding of the ER to the estrogen response element (ERE) within the context of chromatin e.g. within a nucleosome, are largely unknown. To investigate this process, we have reconstituted nucleosomes that contain an ERE located at different translational positions e,g, center, side or edge. Using gel shift assays, we tested whether ER can bind these nucleosomes. We have also found that the non-histone chromatin protein HMOB2 enhances binding of ER to an ERE located at the center of the nucleosome. We determine whether HMGB2 facilitates binding of ER to ERE located at other positions. The results of these studies indicate: 1.) ER binding to the nucleosome is dependent on the location of the ERE. Binding at the edge is better than at the center or side. 2.) Binding of ER is enhanced by HMGB2 regardless of the position of the ERE and at each position, HMGB2-ER-nucleosome complexes can be detected. 3.) Binding of recombinant ER to ERE sites within the nucleosome was greatly reduced compared to ER obtained from nuclear extracts. This suggests that additional proteins contribute to the ability of ER to bind nucleosomes.

SRCAP (SNF2-related CBP activator protein) is a 350-kDa protein that shares homology with the SNF2 family of proteins whose members function in various aspects of transcriptional regulation. In various cell types, SRCAP is found in distinct multiprotein complexes that include proteins found in SWI/SNF chromatin remodeling complexes. SRCAP was identified by its ability to bind to CBP and was found to potentiate the ability of CBP to activate transcription. Studies in our laboratory have demonstrated that SRCAP functions as a coactivator for CREB-mediated transcription of a number of promoters, including that of the phosphoenolpyruvate carboxykinase gene. Our current studies demonstrate that SRCAP enhances phosphoenolpyruvate carboxykinase promoter transcription induced by glucocorticoids. SRCAP also enhances glucocorticoid receptor-mediated transcription of a simple promoter containing only two glucocorticoid response elements, indicating that SRCAP functions as a glucocorticoid receptor coactivator. In similar studies, SRCAP was also found to serve as a coactivator for the androgen receptor. SRCAP exhibits synergistic activation with nuclear receptor coactivators and functionally interacts in vivo with glucocorticoid receptor-interacting protein-1 and coactivator-associated arginine methyltransferase-1. We propose that SRCAP, by virtue of its ability to interact with CBP, functions as a coactivator to regulate transcription initiated by several signaling pathways.

The CBP and p300 co-activators play a key role in many aspects of gene regulation being recruited to the DNA via transcription factors that are targets for specific signaling pathways. It has previously been demonstrated that in neuronal cells the ability of CBP and p300 to activate transcription can be directly stimulated by nerve growth factor or calcium-activated signaling pathways. Here we demonstrate that, in cardiac cells, the activity of CBP and p300 is stimulated by phenylephrine (PE) treatment and that they are required for the activation of atrial naturetic factor (ANF) gene expression by PE. Activation of CBP/p300 by PE involves the p42/p44 MAPK pathway and targets primarily the N terminus of p300 and the C terminus of CBP, which are not homologous to one another. To our knowledge, this is the first report of a specific stimulus modulating the activity of CBP and p300 in cardiac cells and it suggests that these factors play an important role in the hypertrophic effect of PE.

The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Here, we show that activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength–dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras–MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription.

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.

An approach has been optimized permitting measurement of human cytokine reporter gene expression after transient transfection directly into purified human peripheral blood T lymphocytes. Comparing the expression of interleukin 2 (IL-2) CAT with a series of specially engineered gamma interferon (IFN-gamma) constructs, a fundamental difference in the molecular mechanisms regulating these two cytokines has been suggested. A potent, tissue-specific, constitutive-acting positive regulatory element was located between sequences -215 and -53 in the human IFN-gamma gene. Deletion analyses suggested that sequences slightly upstream, between positions -251 to -215, exerted a powerful dominant suppressive influence over that positive element. Negative elements appear to play a major role in controlling the regulation of human IFN-gamma gene expression. We thus propose a model of cytokine gene regulation in which selective derepression may be an important fundamental mechanism of induction and/or positive modulation.

We have used a previously characterized mouse cDNA clone for the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1300-1304), which we designate C alpha, to isolate cDNA clones coding for a second isoform of the C subunit, C beta. C alpha cDNA clones hybridize to a 2.4-kilobase mRNA on Northern blots whereas C beta cDNA clones detect a 4.3-kilobase mRNA. Nucleotide sequence comparison between C alpha and C beta cDNA clones shows that the C beta cDNA codes for a protein which shows 91% identity with C alpha. Determination of mRNA levels for C beta in various tissues shows that it is most highly expressed in brain although it is detectable in all tissues examined. The presence of two genes coding for the C subunit of cAMP-dependent protein kinase may explain past reports of heterogeneity in C subunit protein preparations.

Mouse genes for two isozymes of the catalytic subunit of cAMP-dependent protein kinase (C alpha, C beta) were isolated and characterized. The C alpha gene is divided into 10 exons contained on three overlapping clones. Genomic clones containing the 5' end of the C beta gene were also isolated, and sequence analysis demonstrated that the positions of the three introns examined are identical in the C alpha and C beta genes. S1 mapping and primer extension experiments indicate that transcription initiated from multiple sites in both genes and that neither contain recognizable TATA or CAAT homologies. The 5' flanking regions of both genes are remarkably high in GC content (80% for C alpha and 72% for C beta) and are similar to the "HTF" (HpaII tiny fragment fraction) island sequences which have recently been found to flank several other mammalian transcriptional units.

mRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 amino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using single-stranded RNA probes prepared with SP6 polymerase.

Publisher Summary This chapter focuses on the new approaches to the study of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase system with the utilization of the techniques of molecular genetics. The chapter describes the characterization of two catalytic and four regulatory subunit genes in mammalian systems. Three of these subunits—RIα, RIIα, and Cα—are constitutively expressed in most tissues although the levels of messenger RNA (mRNA) can vary significantly, most notably in male germ cells during spermatogenesis. The other three subunits—Cβ, RIβ, and RIIβ—are highly expressed in brain and are either absent or at very low levels in many other tissues. In particular, the RIIβ gene is highly inducible in several differentiating systems. The chapter further discusses the differences in functional properties of the Cα and Cβ subunits. Future work with specific antibodies should help resolve the potential for specific interactions between subtypes of R and C subunits.

We have previously presented evidence that electrical activity and increased cytosolic calcium reduce the density of sarcolemmal tetrodotoxin (TTX)-sensitive sodium channels in cultured rat muscle cells (Sherman, S. J., and W. A. Catterall (1984) Proc. Natl. Acad. Sci. U. S. A. 81: 262–266). We show here that growth of cells in ryanodine has a biphasic effect on sodium channel number. At low concentrations (0.3 to 10 microM) where this drug releases calcium from the sarcoplasmic reticulum into the cytoplasm, sodium channel number is reduced 62%; whereas, at higher concentrations where total cellular calcium is depleted, the density of sodium channels is increased 40% above control. These results provide further evidence for modulation of sodium channel number by cytosolic calcium. Growth of muscle cells in the presence of agents that mimic cyclic AMP (cAMP) action or increase intracellular cAMP levels including 8-bromo-cyclic AMP (8-BrcAMP), cyclic nucleotide phosphodiesterase inhibitors, and forskolin increased sodium channel density up to 125%. This action did not involve changes in spontaneous electrical activity. Dibutyryl cGMP had no effect. Measurement of the turnover rate of sodium channels after block of channel accumulation by tunicamycin (1.5 micrograms/ml) gave a half- time of 18 hr for exponential decay of TTX-sensitive sodium channels in cultured rat muscle cells after an initial 6-hr lag period. Treatments which modulate sodium channel number through changes in cytosolic calcium or cAMP had no effect on the rate of channel turnover. The increase of sodium channel number after inhibiton of electrical activity or treatment with 8-BrcAMP was half-maximal at 17 hr, consistent with an increase in the rate of sodium channel biosynthesis and/or incorporation into the sarcolemma without a change in channel turnover time. We conclude that cytosolic calcium decreases and cAMP increases sodium channel number by modulating the rate of biosynthesis and/or processing of channel components. The biochemical mechanisms of these regulatory effects are considered.

The catalytic subunit (C) of cAMP-dependent protein kinase holoenzyme type II from bovine cardiac muscle was separated by isoelectric focusing in Immobiline polyacrylamide gels into 9 protein forms. The major forms (i) appeared at pH 7.1, 7.4, 7.5, and 7.7, (ii) exhibited protein kinase activity and were inhibited by heat and acid stable inhibitor, (iii) represented approx. 30%, 4%, 64%, and 1% of the protein respectively, (iv) refocused in the same position from which they had been eluted from the first gel. Antibodies against C detected additional proteins at approx. pH 7.55, 7.75, and 7.8. Two more bands became detectable at approx. pH 7.3 and 7.45 by application of antibody against C β ( Uhler, M.D. & McKnight G.S. 1987, J.Biol.Chem. 262 , 15202–15207 ). The relation of the different forms of C to the fractions C A and C B ( Kinzel V. et al. 1987 Arch. Biochem. Biophys. 253 , 341–349 ) is demonstrated.